A Molecular Dynamics Simulation Study - American Chemical Society

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Interfacial Water Arrangement in the Ice-Bound State of an Antifreeze Protein: A Molecular Dynamics Simulation Study Uday Sankar Midya and Sanjoy Bandyopadhyay* Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur - 721302, India ABSTRACT: Molecular dynamics (MD) simulations have been carried out to study the heterogeneous ice nucleation on modeled peptide surfaces. Simulations show that large peptide surfaces made by TxT (threonine-x-threonine) motifs with the arrangements of threonine (Thr) residues identical to the periodic arrangements of waters on either the basal or prism plane of ice are capable of ice nucleation. Nucleated ice plane is the (0001) basal plane of hexagonal ice (Ih) or (111) plane of cubic ice (Ic). However, due to predefined simulation cell dimensions, the ice growth is only observed on the surface where the Thr residues are arranged like the water arrangement on the basal plane of ice Ih. The γ-methyl and γ-hydroxyl groups of Thr residue are necessary for such ice formation. From this ice nucleation and growth simulation, the interfacial water arrangement in the ice-bound state of Tenebrio molitor antifreeze protein (TmAFP) has been determined. The interfacial water arrangement in the ice-bound state of TmAFP is characterized by five-membered hydrogen bonded rings, where each of the hydroxyl groups of the Thr residues on the ice-binding surface (IBS) of the protein is a ring member. It is found that the water arrangement at the protein-ice interface is distorted from that in bulk ice. Our analysis further reveals that the hydroxyl groups of Thr residues on the IBS of TmAFP form maximum three hydrogen bonds each with the waters in the bound state and methyl groups of Thr residues occupy wider spaces than the normal grooves on the (111) plane of ice Ic. Methyl groups are also located above and along the 3-fold rotational axes of the chair-formed hexagonal hydrogen bonded water rings on the (111) plane.

1. INTRODUCTION The presence of antifreeze proteins (AFPs) in the body fluid of some cold-adopting organisms allows them to survive at subzero temperatures.1,2 AFPs lower the freezing temperature of the body fluid leaving the melting temperature unchanged and thus help the organisms to avoid the possible lethal effect of freezing at low temperatures. This noncolligative lowering of freezing temperature was proposed to occur through adsorption-inhibition mechanism.3 In the presence of seed ice crystal in aqueous solution, AFP molecules irreversibly adsorb on ice surfaces and allow ice to grow on the uncovered surfaces between them. As a consequence, local convex ice−water interfaces develop, and thereby the freezing temperature decreases at the interface according to the Kelvin effect.4 The irreversible nature of the adsorption or binding of the proteins to ice surfaces has been strongly supported by fluorescence microscopy and microfluidic experiments.5,6 The binding process is a peculiar one in the sense that the proteins recognize the solid phase of water, ice, from the large excess of liquid water. For this reason, it is considered the toughest recognition problem in biology.7 Despite significant works during the last 4−5 decades, the driving force of the binding has not been clearly understood. This is mostly due to a lack of experimental AFP−ice complex structure. Computational techniques8−15 and distance based approaches16−20 between the ice waters and that of mostly appeared threonine (Thr) residues on the ice binding surfaces (IBSs) of AFPs have been employed to determine the complex structures. The main focus © 2017 American Chemical Society

in these methods was the positions of hydroxyl and methyl groups of Thr residues with respect to the ice crystal, where the crystallographic arrangement of water molecules was kept unchanged. The ligand of AFP is not a single molecule but a collection of small water molecules connected through weak hydrogen bonds. So, during the binding process any distortion in the water arrangement at the protein−ice interface could be possible from that in ice. Recent solid-state NMR experiment with type III AFP in frozen solution21,22 showed that the interfacial waters on the IBS of the protein remain in ice phase. However, it does not ensure that positions of all these waters should be in ice lattice positions. In the crystal structures of some hyperactive AFPs,16,19,20 it is observed that the IBSs of the proteins contain crystal waters in between an array of Thr residues. The separations between the Thr hydroxyl groups and that between the crystal waters make a good match to the separations of waters in the prism plane of ice. However, the relative arrangement of all the oxygen atoms from Thr hydroxyl groups, which could occupy the ice lattice positions, and the crystal waters closely resembles the water arrangement on the basal plane of ice. This characteristics in the crystal structures indicates that all the interfacial waters probably not occupy the ice lattice positions. So, the water arrangement at the interface is possibly distorted from that in ice. Such distorted Received: April 8, 2017 Published: May 15, 2017 5499

DOI: 10.1021/acs.langmuir.7b01206 Langmuir 2017, 33, 5499−5510

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INPs acts as a template for ice nucleation. As the flat iceinteracting surfaces of the AFPs and INPs are formed by the same motifs, it was suggested30,31 that the large difference in the size of the active surfaces of AFPs and INPs is responsible for their opposite effect on ice growth. Due to larger size of the active surfaces of INPs, they catalyze ice formation, and due to smaller size of the active surfaces of AFPs, they could not nucleate ice but bind to ice surfaces to stop the ice growth. This hypothesis was supported by the experimental results of Tsuda and co-workers.32 They showed that a 96-residue segment of the INP from the bacteria Pseudomonas syringae exhibits moderate antifreeze activity. The hydration waters on the IBSs of AFPs have shown to be ice-like in character.33−35 Antifreeze activity of a small segment of the INP therefore indicates ice-like characteristics of the hydration waters on the templating surfaces. Remember that both surfaces are formed by the same TxT motifs and have the same tertiary structures except size. Due to the larger size of INPs, the size of the icelike hydration water is large. This large ice-like hydration water accelerates the ice formation. The characteristics of the hydration waters and the structural similarities in the active surfaces of INPs and AFPs also indicate that the protein−ice interfacial water arrangements in the ice-bound state of both types of proteins will be similar across the interfaces. Moreover, the mechanism of the ice nucleation activity of INPs itself suggests that a large modeled peptide surface made with the periodic arrangement of TxT motifs and by maintaining the same flatness nature as in the proteins should catalyze heterogeneous ice nucleation. If the modeled surface catalyzes ice formation, the surface−ice interfacial water arrangement will be similar to that at protein−ice interfaces. Thus, heterogeneous ice nucleation on a modeled peptide surface could be used as a tool for the determination of the AFP−ice interfacial water arrangement. Our objectives are to simulate ice formation on such modeled peptide surfaces and to determine, from a successful simulation, the interfacial water arrangement in the ice-bound state of hyperactive AFPs like the TmAFP considered here. The rest of the article has been organized as follows: In Section 2, we provide a brief description of the system setups and the simulation methods employed. The results obtained from our analyses are presented and discussed in the following section (Section 3). Finally, the important findings from the study and the conclusions reached therefrom are highlighted in Section 4.

arrangement, if any, is needed to be found out in order to obtain AFP−ice complex structure. Ice nucleating proteins (INPs) found in some bacterial cell membranes23 behave seemingly opposite to that of AFPs. INPs impede the supercooling of liquid water by acting as a template for heterogeneous ice nucleation. Droplet of pure water could be supercooled down to its homogeneous ice nucleation temperature, − 41 °C, at atmospheric pressure.24 However, INPs allow water to freeze at a temperature as high as −2 °C.25,26 Apparently INPs have an opposite affect on the ice growth relative to that by AFPs.27 AFPs stop the ice growth upon binding to ice surfaces, whereas INPs catalyze ice formation. The catalytic activity of INPs resides on the large repetitive central domains of the proteins.23 The central domains of INPs are structurally similar to the hyperactive AFPs from Tenebrio molitor (TmAFP) and spruce budworm (sbwAFP). In the primary sequences of the central domains and of the AFPs, the TxT (threonine-x-threonine) motif appears periodically. Primary sequences of TmAFP28 and sbwAFP29 consist of four to six TxT motifs in a period of 12 and 15 residues, respectively (Figure 1). On the other hand, the

Figure 1. Crystal structures of the (a) TmAFP protein16 and (b) sbwAFP protein17 (drawn as cartoons). IBSs of the proteins are drawn in blue, while the remaining parts are drawn in green. Side chains of the two sets of threonine (T) residues in the IBSs are drawn as sticks. The primary amino acid sequences of the proteins (in one-letter code) are indicated below the respective structures.

2. SYSTEM SETUPS AND SIMULATION DETAILS We have prepared four peptide surfaces named T-basal, Tprism, S-basal, and S-prism. T-basal and T-prism surfaces were made with Thr and Gly (glycine) residues, whereas S-basal and S-prism surfaces were made with Ser (serine) and Gly residues. The words “basal” and “prism” in the names are used to emphasize that the separations between the Thr or Ser residues in the surfaces mimic the periodic separations between the water molecules on the basal (7.83 and 4.52 Å) and prism (7.35 and 4.52 Å) plane of ice, respectively. We have placed the Thr residues with this two types of periodic separations because, in hyperactive AFPs16,17,20 and in INPs,30,31 Thr residues nearly mimic these two separations on a flat surface. Initially the coordinates of a TCT (threonine-cysteine-threonine) motif were taken from the crystal structure of TmAFP16 (PDB code: 1EZG). The x residue of TxT motifs point inward to the core of AFPs.16,17,20 Therefore, its side chain has little role on the activity of the proteins. For simplicity, we have converted the

central domains of INPs contain ∼60 TxT motifs in a period of 16 residues.23 Although the higher order structures of the central domain have not been determined so far by experiment, the molecular modeling studies30,31 indicate a long β-helical structure of the central domain with 16 residues in each helical loop. TxT motifs form a flat surface on one side of the helix. This flat surface is the active surface of the INP. TmAFP and sbwAFP are also β-helical, with the TxT motifs forming a flat surface on one side of the proteins16,17 (Figure 1). Active surface of each of the AFPs is also the flat surface. However, active surfaces of INPs are larger in size than the active surfaces of TmAFP and sbwAFP, as the INPs contain ∼60 TxT motifs on the active surfaces compared to four to six TxT motifs in the AFPs. Active surface of each of the AFPs binds to ice surfaces to stop the ice growth, whereas, the active surface of each of 5500


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gradually heated to 300 K within short runs of 100 ps each and equilibrated for 2 ns each under isothermal−isobaric ensemble (NPT) conditions at a constant pressure of 1 atm. Finally each of the systems was cooled down to 220 K and continued the simulation at that temperature for 100 ns under NPT condition at a constant pressure of 1 atm. Backbone heavy atoms of the peptides were kept frozen during all the simulations. Temperature of the systems was controlled by the Langevin dynamics method with a friction constant of 1 ps−1, while the pressure was controlled by the Nosé−Hoover Langevin piston method.37 During the simulations, systems were allowed to fluctuate only along the z-direction. The simulations were carried out with an integration time step of 1 fs. All bonds involving the hydrogen atoms were constrained by the SHAKE algorithm.38 The periodic boundary conditions and the minimum image convention39 were employed to calculate the short-range Lennard-Jones interactions with a spherical cutoff distance of 12 Å and a switch distance of 10 Å. The long-range electrostatic interactions were calculated using the particlemesh Ewald (PME) method.40 The all-atom CHARMM22 force field and potential parameters for peptides with CMAP corrections41,42 and TIP4P model43 for water were employed in the calculations. The simulated temperature, 220 K, employed here is well below the melting temperature of TIP4P water, 232 K.44 However, the CHARMM force field was parametrized near room temperature, and such low temperature simulations could model inaccurate peptide−water as well as inter- and intrapeptide interactions. Here the inter- and intrapeptide interactions are not so important as the backbone was kept frozen during the simulation. However, the peptide−water interaction is crucial and the subject of verification for accurate modeling. For that purpose, we repeated the simulation of the system containing T-basal surface at higher temperature, 260 K, using a TIP4P/Ice45 water model and obtained exactly the same results as with the TIP4P water at 220 K temperature, indicating the persistence of accurate peptide−water interaction at 220 K temperature.

inward pointed cysteine residue of the TCT motif to glycine residue by deleting the side chain coordinates of the cysteine residue. The coordinate of Cα atom of the first Thr residue was set to the origin of the Cartesian coordinate system, and the resulting TGT (threonine-glycine-threonine) motif was rotated to align the Cα−Cβ bond of the first Thr residue along the zaxis of the coordinate system. In the TGT motif, the angle between the aforementioned bond and the vector connecting the Cα atoms of the two Thr residues was 89.7 °C. So, the vector can be considered to be aligned along the x-axis of the Cartesian coordinate system. The last Thr residue of the TGT motif was deleted, and to make a peptide chain of the sequence TGTGTGTGTGTGTGTGTGTG, the resulting TG motif was replicated 10 times along the x-axis with a separation of either 7.83 Å (for T-basal surface) or 7.35 Å (for T-prism surface) between the successive Thr residues. Construction of each of the T-basal and T-prism surfaces was completed by replicating the peptide chain 13 times along the y-axis of the coordinate system with a separation of 4.52 Å between the successive chains. S-basal and S-prism surfaces were constructed just by deleting the γ-methyl groups of the Thr residues in the T-basal and T-prism surfaces, respectively. These two surfaces are considered here to check whether the γ-methyl group of Thr is required for ice nucleation or not. T-basal and S-basal surfaces are shown in Figure 2.

Figure 2. Structures of the (a) T-basal surface and (b) S-basal surface. Backbones of the peptide chains are drawn in green. Side chains of threonine (Thr) residues and serine (Ser) residues are drawn as sticks. For convenience, a plane (colored green) is drawn in each surface.

3. RESULTS AND DISCUSSION 3.1. Structure and Ordering of Surface Water. Several simulation studies46−48 suggest that ordering of hydration water is important for a substrate to be an effective ice nucleator. Structural behavior of the hydration water is therefore needed to be examined in order to assess the ability of the surfaces for ice nucleation. Such aspect is explored in this section. For our purpose, we are only interested on the Thr or Ser projected side of the surfaces, which is why we analyzed only that side of the surfaces. The results presented in this section are obtained by averaging over the last 25 ns trajectory of each of the systems. Structural arrangements of water molecules on the peptide surfaces have been explored by calculating the pairwise correlation function, s(r), between waters and the nonhydrogen atoms of the surfaces. In the calculation of s(r), minimum distance criteria are used to identify the tagged water relative to a non-hydrogen atom of a surface. In addition, the s(r) values are normalized by using the actual volume available to water within a shell at a distance r with thickness δr instead of by the entire volume of the corresponding spherical shell, as usually done in standard radial distribution function (rdf) calculations. We have employed the method proposed by Astley et al.49 to calculate the available volume of water around the peptide surfaces. s(r) may be regarded as the surface

We have carried out four simulations taking one peptide surface in each case. The simulations were performed using the NAMD package.36 To adjust the backbone bond lengths, each of the surfaces was energy minimized separately in gas phase with frozen side chain heavy atoms followed by another minimization for each surface with frozen backbone heavy atoms. Conjugate gradient energy minimization method as implemented in the NAMD code36 was used for the minimizations. Preparation of the systems were completed by inserting the energy minimized surfaces separately into preequilibrated water boxes. In this process, water molecules that were within 2 Å from any peptide atom were removed. The dimensions of the systems were chosen in such a way that a surface could extend across the periodic boundary to make an infinitely large surface. With this criteria, the dimensions of the selected orthorhombic cells containing T-basal, T-prism, Sbasal, and S-prism surfaces were taken as 78.3 Å × 58.76 Å × 60.00 Å, 73.5 Å × 58.76 Å × 60.00 Å, 78.3 Å × 58.76 Å × 60.00 Å, and 73.5 Å × 58.76 Å × 50.00 Å, respectively. Further minimizations were done for all systems, keeping all the peptide heavy atoms frozen in their positions. The systems were then 5501


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the surface. The order parameter distribution, P(qt), for first hydration shell water molecules (which are within 5 Å from the non-hydrogen atoms) has been calculated for each of the peptide surfaces. The results are shown in Figure 3b. For comparison, the corresponding distributions for pure bulk water and for pure bulk ice as obtained from two separate simulations at the same temperature are included in the figure. It is clearly evident that the local tetrahedral ordering of waters on T-basal and T-prism surfaces is higher than that on S-basal and S-prism surfaces. The local ordering of waters on S-basal and S-prism surfaces much more resembles that in bulk water. By contrast, the tetrahedral ordering of waters on T-basal and T-prism surfaces is ice-like, as the corresponding distributions are peaked around the same position as that in bulk ice. The ice-like local environments of the water molecules well correlate with the long-range water structuring on those surfaces. Next, we calculated the distribution of minimum angle out of four possible ∠H−O···O angles formed between the two hydration shell water molecules separated by less than 4 Å. This is a bimodal distribution, and could be used as a tool to identify the hydrophobic/hydrophilic nature of a surface.53,54 Compared to bulk water, greater low-angle population for hydration waters indicates the hydrophobic nature of a surface. Moreover, the greater low-angle population indicates greater fraction of hydrogen-bonded water pairs around a surface than in bulk phase. The distributions for the hydration waters on the four peptide surfaces along with that of pure bulk water and bulk ice are depicted in Figure 3c. From the figure it is obvious that the fraction of hydrogen-bonded water pairs on T-basal and Tprism surfaces is significantly higher than that in bulk water as well as that on the S-basal and S-prism surfaces. However, the fraction is low compared to bulk ice. The greater low-angle population of waters on Thr-based surfaces than that on Serbased surfaces correlates well with the relative hydrophobic nature of these two residues. The above calculations demonstrate that hydration waters on T-basal and T-prism surfaces exhibit structurally different behaviors than that on Sbasal and S-prism surfaces. Importantly, hydration waters on Tbasal and T-prism surfaces are ice-like and strongly structured up to longer distance from the surfaces, which clearly indicates their ability for ice nucleation. 3.2. Nucleation and Growth of Ice on the Surfaces. In order to identify the nucleation and growth of ice, if any, on peptide surfaces, a suitable order parameter is required to distinguish ice-like water molecules from the liquid-like water molecules. One such order parameter, ⟨q6 ⟩, is derived based on Steinhardt order parameters55 and defined as56,57

distribution function of waters, as the employed minimum distance criteria measures the distance of waters from microscopically ragged peptide surfaces. Results obtained for the four surfaces are shown in Figure 3a. Strong water

Figure 3. (a) Surface pair correlations function, s(r), of water molecules as a function of distance from the non-hydrogen atoms of the peptide surfaces. (b) Distribution of the tetrahedral order parameter, P(qt), for water molecules that are present within 5 Å from the non-hydrogen atoms of the surfaces. Corresponding distributions for pure bulk water and bulk ice are included for comparison. (c) Distributions of minimum angle among four possible ∠H−O···O angles formed by any two waters that are separated from each other by less than 4 Å, and each presents within 5 Å from the non-hydrogen atoms of the surfaces. The corresponding distributions for pure bulk water and bulk ice are also included for comparison. Measured angle is depicted inside the figure. The Thr/Ser projected side of the surfaces has only been considered for the above calculations.

structuring with several well-defined solvation shells extending to longer distance on the Thr-based surfaces can be easily seen from the figure. The water distribution is even sharp on the Tbasal surface. This is a clear indication of ice formation on that surface. Importantly, water structurings within the first hydration shell (within 5 Å) on T-basal and T-prism surfaces are similar. Water structuring on the Ser-based surfaces is not as prominent as that on the Thr-based surfaces. S-basal and Sprism surfaces exhibit almost identical water structuring, which extends only up to the first hydration shell. The result demonstrates that, compared to Ser residues, periodically arranged Thr residues on flat surfaces strongly influence the nearby water molecules in such a way that the waters align in an ordered manner over a long distance from the surface. Local environments of water molecules near the peptide surfaces have been explored by calculating the tetrahedral order parameter, qt, defined as50−52 qt = 1 −

3 8

3

⎡ 4π ⟨q6(i)⟩ = ⎢ ⎣⎢ 13

⟨q6m(i)⟩ =

∑ ∑ j=1

∑

2⎥

|⟨q6m(i)⟩|

m =−6

⎥⎦

(2)

where

4

2 ⎛ 1⎞ ⎜cos ψ + ⎟ jk ⎝ 3⎠ k=j+1

⎤1/2

6

1 Nng + 1

Nng

∑ q6m(j) j=0

(3)

and

(1)

where, ψjk is the angle between the bond vectors rij and rik, where j and k are the four atoms that are nearest neighbors to the ith water. The calculations are carried out by assuming that the non-hydrogen atoms of a peptide surface can act as neighboring sites for the first layer of water molecules around

1 q6m(i) = Nng

Nng

∑ Y6m(θij , φij) j=1

(4)

Y6m(θij,φij) is the sixth-order spherical harmonics, θij and φij are the polar angles of the bond vector connecting ith water 5502


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Langmuir molecule to the jth member of its Nng number of neighboring molecules. Angles are measured in an external reference frame. Summation in eq 3 runs over all the neighbors of i water molecule, including the molecule itself (when j = 0). For better discrimination between ice-like water and liquid-like water molecules, we have considered only second-shell neighbors of a tagged water molecule58 in the calculation. This order parameter has previously been used for such purpose.56,57 Distribution of ⟨q6⟩, P(⟨q6⟩), for bulk ice water, bulk liquid water, and the system coexisting with the two phases is shown in Figure 4. The distribution of bulk ice phase is well separated

surfaces has been depicted. It is evident that a significant fraction of ice water molecules appears on the T-prism surface. However, the fraction does not increase with time, indicating only the occurrence of ice nucleation on that surface. The picture further suggests that no ice water molecule appears on the S-basal and S-prism surfaces. Structural behavior of hydration waters also exhibit the absence of ice-like water structure on these two surfaces. Certainly, the Ser residues periodically arranged like the waters on the basal or prism plane of ice on a flat surface do not nucleate ice. However, the Thr residues with similar arrangements on a flat surface nucleate ice. Figures 6 and 7 show a few snapshots of the systems containing T-basal and T-prism surfaces, respectively. From the figures it is evident that nucleation and subsequent growth of ice occur on the T-basal surface, but only nucleation of ice occurs on the T-prism surface. Nucleation occurs only on the Thr-projected side of the surfaces. We have examined the hydrogen bonding pattern of the nearby water molecules with the peptide chains of the surfaces. Interestingly, it is observed that waters form hydrogen bonds extensively with the hydroxyl groups of Thr residues but very negligibly with the potential hydrogen bond forming backbone atoms. This suggests that nucleation of ice is largely influenced by the side chain (1hydroxyethyl group) of the Thr residues and backbone atoms has little role on it, indicating the potential of ice nucleation of 1-hydroxyethyl groups arranged in either of the two fashions on any flat surface. We define the interfacial (ice) waters of the two surfaces as the water molecules located on the Thr-projected side of the surfaces and reside within 5 Å from the nonhydrogen peptide atoms. These waters are indicated within the blue-dashed boxes in Figures 6b and 7b. To understand the spatial structuring of the interfacial water molecules, we have computed their two-dimensional density distributions on the plane parallel to the peptide surface. Results are presented in Figure 8a,b. As expected, the distributions are characterized by high intensity peaks. But more importantly, hexagonal arrangement of peak positions appears on the distributions. Offhexagonal peaks are also present, and these originate from the interfacial waters located on the plane formed by the side chains of the Thr residues. To confirm that, we repeated the same calculation with those interfacial waters molecules that are situated above the side chains. The results are displayed in Figure 8c,d. Clearly off-hexagonal peaks are absent in the distributions. The calculation reveals that the interfacial waters have a hexagonal arrangement on the plane parallel to peptide surface. Interestingly, the arrangements are identical on the Tbasal and T-prism surfaces. Water molecules on the basal plane of ice are also identically arranged (Figure 8e). Therefore, Tbasal and T-prism surfaces nucleate basal plane of ice. Waters in the nucleated basal plane on T-prism surface are squeezed with respect to that on the T-basal surface because of the presence of same number of water molecules (520 waters) in the nucleated basal plane on the smaller T-prism surface (surface area 4318 Å2 as compared to 4600 Å2 that of T-basal surface). The strong peptide−water interactions make this squeezing possible. As the dimension of the T-basal surface is matched to the basal plane of ice, the water molecules in the nucleated basal plane on that surface are not squeezed with respect to waters in the pure ice. In other words, the nucleated basal plane is in crystallograpic constraint on the T-prism surface but not on the T-basal surface. As a result, ice grows continuously on the Tbasal surface (Figure 6c,d) but not on the T-prism surface (Figure 7c,d), leading to only ice nucleation on the later

Figure 4. ⟨q6 ⟩ order parameter distributions, P(⟨q6 ⟩), for water molecules in pure bulk liquid phase, pure bulk ice phase, and in a lquid−ice coexisting system.

from that of bulk liquid phase with no overlap between the two distributions. In a coexisting system, a fraction of population appears in between the distributions of two bulk phases. This population corresponds to ice−water interfacial water molecules, which are either quasi-ice-like or quasi-liquid-like. A cutoff, 0.17, for ⟨q6⟩ is used to identify a water molecule as an ice water or nonice water (interfacial and liquid water). A water molecule having ⟨q6⟩ value greater than the cutoff is an ice water molecule. Variation of fraction of ice water molecules on the four peptide surfaces has been displayed in Figure 5. Increase in the fraction of ice water molecules with time on the T-basal surface ensures the nucleation and growth of ice on that surface. In the inset of the figure, an enlarged view of the variation of ice fraction with time on the remaining three

Figure 5. Time evolutions of the fraction of ice water molecules, f ice, formed on the T-basal, T-prism, S-basal, and S-prism surfaces. Enlarged view of the evolution for the last three surfaces (T-prism, Sbasal, and S-prism) is drawn in the inset. 5503


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Figure 6. Snapshots of a few representative water arrangements on the T-basal surface as obtained at (a) 0 ns, (b) 25 ns, (c) 50 ns, and (d) 100 ns. Peptide surface is drawn as sticks. Water molecules are drawn as balls and sticks. The waters within the blue-dashed box drawn in panel b are defined as the interfacial water of the surface.

Figure 7. Snapshots of a few representative water arrangements on the T-prism surface as obtained at (a) 0 ns, (b) 25 ns, (c) 50 ns, and (d) 100 ns. Drawing scheme is the same as that in Figure 6. The waters within the blue-dashed box drawn in panel b are defined as the interfacial water of the surface.

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Figure 8. Two dimensional density distributions of all the interfacial water molecules on (a) T-basal and (b) T-prism surfaces. The corresponding distributions excluding the water molecules residing on the plane formed by the hydroxyl groups of Thr residues on (c) T-basal and (d) T-prism surfaces. White lines connect the hexagonally arranged peaks. (e) Arrangement of water molecules on the basal plane of ice Ih. The red spheres represent the oxygens of the water molecules on that plane.

surface. These results suggest that the Thr residues arranged on a surface with close resemblence of the water arrangements on the basal or prism plane of ice order the water molecules in very specific arrangements like the waters on the basal plane of ice. Such ordering has also been reported in our early study with TmAFP59 and correlates well with the preferential binding of hyperactive AFPs60−62 and INP63 to the basal plane of ice. Recall that the active surface of these proteins are made by TxT motifs. Figure 6c,d shows that, although the T-basal surface nucleates the basal plane of hexagonal ice, the formed ice is not the stable hexagonal ice (Ih), but metastable cubic ice (Ic). Actually, the water arrangement on a (0001) basal plane of hexagonal ice is identical to that on a (111) plane of cubic ice. The nucleated ice plane may therefore be regarded as either the (0001) basal plane of hexagonal ice or the (111) plane of cubic ice, suggesting the possibility of formation of either of the two polymorphs of ice. From supercooled water, cubic ice prefers to grow over the hexagonal ice64,65 because of the tendency of more compact local structure formation on the growing ice front.66 Previously this preference has been attributed to the lower surface tension of the cubic ice compared to hexagonal ice.67,68

Figures 9 and 10 show a few snapshots of the systems containing S-basal and S-prism surfaces, respectively. From the figures it is evident that no ice nucleation occurs on those surfaces. Thr and Ser residues are different with respect to the presence of a γ-methyl group. The γ-methyl group is present in Thr residue, whereas it is absent in Ser residue. The absence of γ-methyl group in Ser residue allows it to change its rotameric state quite frequently during the course of simulations. This change may cause the surfaces to be ineffective for ice nucleation. To explore such possibility, we separately carried out another 50 ns simulation with the S-basal surface keeping all the peptide heavy atoms frozen in their initial positions. We observed no ice nucleation in that simulation. The results therefore suggest that the γ-methyl group of Thr is essential for ice nucleation. Not only the γ-methyl group, but the γ-hydroxyl group is also important for ice nucleation, as we observed no ice nucleation in an another 100 ns simulation with a surface made by valine (Val) residues in place of Thr residues in the Tbasal surface under the same conditions. Val residue contains two γ-methyl groups but not a γ-hydroxyl group. Inefficiency of ice nucleation by the Ser and Val residues indicates the absence of ice-like structuring of hydration waters on the surfaces 5505


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Figure 9. Snapshots of a few representative water arrangements on the S-basal surface as obtained at (a) 0 ns, (b) 25 ns, (c) 50 ns, and (d) 100 ns. Drawing scheme is the same as that in Figure 6.

Figure 10. Snapshots of a few representative water arrangements on the S-prism surface as obtained at (a) 0 ns, (b) 25 ns, (c) 50 ns, and (d) 100 ns. Drawing scheme is the same as that in Figure 6.

residues or Val residues,13,69−72 is the possible reason for their reduced antifreeze activity. Several simulation studies46−48,73−78 have been attempted for heterogeneous ice nucleation. However, only a few were

formed by them. As ice-like hydration structure has been proposed to be involved in the binding mechanism of AFPs to ice,33−35,59 lack of such structuring in some mutants, where Thr residues in active surface have been replaced by either Ser 5506


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Langmuir successful,46,48,76,77 whereas others47,73−75,78 ended with the formation of one or two adlayers near the surfaces, failing in subsequent ice formation. It was observed that a good lattice match, according to the earlier theory of heterogeneous ice nucleation,79 between the active sites of the templating surface and the waters on a ice plane is not sufficient for ice nucleation.73,78,80,81 Indeed, structuring of interfacial water by the surface as a whole is crucial for such an event to occur.46−48 If structuring of interfacial water matches with the water arrangement on a ice plane, ice will form. Here the structuring of the interfacial waters on T-basal and T-prism surfaces matches with the water arrangement on the (0001) basal plane of hexagonal ice or (111) plane of cubic ice. Although the Thr hydroxyl groups, which can form hydrogen bonds with waters, on the T-prism surface match with water positions on the prism plane of ice, it does not nucleate the ice prism plane, but rather nucleates the ice basal plane. However, due to constraint imposed by the predefined cell dimension, ice does not grow on that surface at the simulated temperature. On the other hand, cell dimension of the system containing T-basal surface has been matched with the ice basal plane. Therefore, such constraint is absent, and ice grows on that surface. In the case of Ser- or Val-based surfaces, structured interfacial waters are not formed, and hence ice formation is not seen. These results clearly indicate that the TxT motifs are capable of heterogeneous ice nucleation when they form a large surface. However, in small surfaces like the IBS of AFPs, they do not nucleate ice. Remember that, in our previous study, we did not see any ice formation at the IBS of TmAFP at the same temperature.59 The large surfaces we made with TxT motifs in this study perhaps has no existence in a real system. However, the surfaces could be considered quite analogous to the active surface of INPs. In the INPs, only one TxT motif appears per helical loop, and ∼60 such loops exist. In addition, the INP molecules laterally associate to form polymeric structure and thus extend the active surface perpendicular to the helical axis of the protein.30,31 3.3. Protein-Ice Interfacial Water Arrangement. In the Introduction, we discussed that due to the presence of TxT motifs on the IBS of TmAFP and sbwAFP, on the active surface of INPs and on simulated Thr-based surfaces, the interfacial water arrangements will be the same in the ice-bound state of the surfaces. The ice formed on the T-basal plane could therefore be mapped on the IBS of either of the AFPs in order to get AFP−ice interfacial water arrangement. Here we have considered the TmAFP. We have mapped four central strands in the IBS of TmAFP on a part of the T-basal surface with same number of Thr residues as in the four strands. The final configuration of the T-basal surface was chosen to get the nucleated ice on it. After the mapping, the T-basal surface and the water molecules present opposite to the Thr-projected side of the surface were discarded. Now the water molecules are positioned with respect to the mapped IBS. The result is shown in Figure 11a. The protein−ice interfacial water arrangement is clearly evident from the figure. In solution there exist five sets of order water molecules on the IBS of TmAFP. 59 Corresponding waters are also shown here by labeling as a to e. The appearance of these sets of waters indicates that nature of the hydration waters on the large model surface and on the small IBS are similar. The interfacial water arrangement is characterized by the five-membered hydrogen bonded rings (Figure 11b). This is an important finding as there exist no fivemembered hydrogen bonded ring in normal ice, but the ring

Figure 11. (a) Ice-bound state of TmAFP. Five sets of water molecules that appeared in the solution of TmAFP59 are colored differently and marked as a−e. Whole protein is drawn as cartoons, and the residues on IBS are also separately drawn as sticks. (b) Hydrogen bonding pattern of a Thr residue on the IBS with the nearby water molecules in bound state. Hydrogen bonds are drawn as red dotted lines. (c) Position of the γ-methyl group of a Thr residue with respect to the hexagonal hydrogen-bonded water ring in ice. Water molecules are drawn as balls and sticks.

appears to be involved in the attachment of the AFP with ice. Such five-membered rings in the protein−ice bound structure had not been proposed previously, but were found to exist in the crystal structure of bacterial AFP.19 Thr hydroxyls and the set a and b waters remain in a plane, and the other three sets of waters (c, d, and e) remain in a plane that is parallel to the first plane. Set a and b waters correspond to the off-hexagonal peaks in Figure 8a,b. The sets c, d, and e actually belong to the (111) plane of the nucleated ice. Obviously they occupy the ice lattice positions. However, the set a and b waters do not occupy the ice lattice positions as evident from the figure. The interfacial water arrangement is therefore distorted from that in bulk ice. The number of hydrogen bonds formed by a Thr hydroxyl group is three (Figure 11b,c), the maximum value that was thought to be formed in the binding configuration where all the interfacial waters as well as hydroxyls groups of Thr occupy ice lattice positions.82 A hydroxyl group is a member of three hydrogen-bonded rings: two five-membered and one sixmembered (Figure 11b). The six-membered ring is in boat form. This ring is not perpendicular to the (111) plane. As a result, a wider space than the groove in the (111) plane of ice Ic is created, and the methyl group of the Thr side chain is accommodated there. Each of the methyl groups of Thr residues is positioned above and, along the 3-fold rotational axis of the chair-formed hexagonal water rings in the ice plane (Figure 11c). The interfacial water arrangement and hence the protein−ice bound structure derived here is different from those where the water arrangement in ice was kept intact.8−20 In these structures, either binding energy8−15 or hydrogen bond formation16−20 has been shown to be favorable. In a real system, no sharp boundary exist between the ice and liquid water. AFPs bind to this diffused ice−water interface. Its binding is not like the normal receptor−ligand binding, where the active surface of the receptor and ligand are sharp and “visible” to each other. Instead the AFPs bind to ice through the merging of two interfacial water layersone from the IBS of 5507


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with waters. For the interfacial waters, the arrangement is found to be distorted from that in bulk ice, and therefore may be regarded as the clathrate structure. This clathrate structure is formed by both the hydrophobic and hydrogen bonding effect of the Thr residues. In the ice-bound state, the methyl group of Thr residues occupy a wider space than the normal groove in the ice plane, and each is located along the 3-fold rotational axis of chair-formed hexagonal water rings on the (111) plane. Since the positions of the waters at the interface is solely guided by the Thr residues, the presented interfacial water arrangement may be regarded as the suitable one that is likely to exist in the ice-bound state of TmAFP. This arrangement could be used in the future to calculate the driving force for the binding of AFPs to ice.

AFP and the other from the icefollowed by the freezing of the merged zone.33 During this freezing, all the waters in the merged zone can not occupy the ice lattice positions, as the waters are under the influence of AFP as well as ice. So, the arrangement of the waters in the merged zone, which we have defined as the protein−ice interface is distorted from that in bulk ice. This type of interfacial arrangement on hyperactive bacterial AFP has been categorized as clathrate structure.19 Importantly, the (111) plane of ice Ic is present in the clathrate structure derived here. The very specific clathrate structure is formed by hydrophobic interaction as well as hydrophilic interaction, as we observe no such structure formation on Ser (hydrophilic) surfaces and on the Val (hydrophobic) surface. Such structure is actually formed by both the hydrophobic effect of the γ-methyl group and the hydrogen bonding (hydrophilic) effect of the γ-hydroxyl group of Thr residues.19,70 Waters around hydrophobic solutes are generally more structured with enhanced tetrahedral environment compared to bulk.83−86 However, to integrate this locally clathrate-like environment around each of the γ-methyl groups into a particular ice-plane-like structure, a directive group is required. The γ-hydroxyl group of Thr residues on the IBS plays that directive role. Thr amino acid is unique in that respect, which contains both hydrophobic (methyl) and hydrophilic (hydroxyl) groups. That is probably why nature choose it to form most of the active surfaces of AFPs and INPs. This type of dual role of Thr residues has also been observed in the binding mechanism of AFPs to methane clathrate hydrate ice-like surfaces.87 Apparently, this dual role can be correlated to reduced antifreeze activity of the AFPs on deletion of either of the two groups or both.13,69−72

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the Department of Science and Technology (DST), Government of India, under the DST-FIST programme and the DST-IYC award. U.S.M. thanks the Council for Scientific and Industrial Research (CSIR), New Delhi, for providing a scholarship.

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REFERENCES

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4. CONCLUSIONS In this report, we have carried out atomsitic MD simulations of model peptide surfaces at 220 K temperature. Efforts have been made to simulate heterogeneous ice nucleation on the surfaces made by TxT motifs. Alongside, an attempt has also been made to demonstrate the interfacial water arrangement, extracted from a successful simulation of heterogeneous ice nucleation, in the ice-bound state of TmAFP. Simulations show that peptide surfaces made with Thr residues mimicking the periodic separations of water molecules on either the basal plane or prism plane of ice are capable of ice nucleation. The nucleated plane is the (111) plane of ice Ic or the (0001) plane of ice Ih. Ice growth was observed when the simulation box dimension was matched with the (111) plane dimension of ice Ic. ((0001) plane of ice Ih and (111) plane of Ic are identical.) That matching was present when the Thr residues on a surface were arranged like the water arrangement on the basal plane of ice Ih. Simulations with structurally similar peptide surfaces made separately with Ser or Val residues, instead of Thr residues, indicate that the γ-methyl group and γhydroxyl of Thr is required for the ice nucleation. The results support that the molecular origin of the differential activity of INPs and AFPs is their difference in size of the active surfaces. Large active surfaces made with TxT motifs in INPs act as a template for the heterogeneous ice nucleation. Smaller IBSs made with similar TxT motifs could not nucleate ice but binds to the ice for the inhibition of further ice growth. Water arrangement at the IBS of TmAFP in ice-bound state is characterized by five-membered hydrogen bonded rings, where the Thr hydroxyl group is a ring member. The hydroxyl group forms three hydrogen bonds, the maximum it can form 5508


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